Medizin - Open Access LMU - Teil 20/22 Archiv

Background: Nasal colonization with methicillin-resistant Staphylococcus aureus (MRSA) is a well defined risk factor for subsequent bacteremia and death in various groups of patients, but its impact on outcome in patients receiving long-term hemodialysis (HD) is under debate. Methods: This prospective interventional cohort study (performed 2004 to 2010) enrolled 289 HD outpatients of an urban dialysis-unit. Nasal swab cultures for MRSA were performed in all patients upon first admission, at transfer from another dialysis facility or readmission after hospitalisation. Nasal MRSA carriers were treated in a separate ward and received mupirocin nasal ointment. Concomitant extra-nasal MRSA colonization was treated with 0.2% chlorhexidine mouth rinse (throat) or octenidine dihydrochloride containing antiseptic soaps and 2% chlorhexidine body washes (skin). Clinical data and outcome of carriers and noncarriers were systematically analyzed. Results: The screening approach identified 34 nasal MRSA carriers (11.7%). Extra-nasal MRSA colonization was observed in 11/34 (32%) nasal MRSA carriers. History of malignancy and an increased Charlson Comorbidity Index were significant predictors for nasal MRSA carriers, whereas traditional risk factors for MRSA colonization or markers of inflammation or malnutrition were not able to discriminate. Kaplan-Meier analysis demonstrated significant survival differences between MRSA carriers and noncarriers. Mupirocin ointment persistently eliminated nasal MRSA colonization in 26/34 (73.5%) patients. Persistent nasal MRSA carriers with failure of this eradication approach had an extremely poor prognosis with an all-cause mortality rate >85%. Conclusions: Nasal MRSA carriage with failure of mupirocin decolonization was associated with increased mortality despite a lack of overt clinical signs of infection. Further studies are needed to demonstrate whether nasal MRSA colonization represents a novel predictor of worse outcome or just another surrogate marker of the burden of comorbid diseases leading to fatal outcome in HD patients.

Background: To identify dose-volume parameters predictive for severity of acute esophagitis (CTC > grade 2) in locally-advanced non small-cell lung cancer (LA-NSCLC) patients treated with neoadjuvant concurrent hyperfractionated-accelerated chemoradiotherapy (HA-CRT) a retrospective analysis was performed. 88 patients were treated with HA-CRT followed by radical surgery. Predictive power of absolute oesophageal length, absolute and relative oesophageal volume included in the 95\%-isodose, patient-and tumor-related factors for severity of acute esophagitis was assessed. Findings: A total of 82 patients (93\%) developed radiation-induced acute esophagitis. Grade 1 was documented in 1 (1\%), grade 2 in 55 (67\%), grade 3 in 23 (28\%) and grade 4 in 3 (4\%) patients, respectively. Absolute oesophageal volume included in the 95\%-isodose (42.8 Gy) achieved 13.5 cm(3) (range: 3 - 29 cm(3)). Of the tested variables in univariate analysis, absolute oesophageal volume included in the 95\%-Isodose was found to be the only significant variable (p = 0.03) predicting severe acute esophagitis (CTC > grade 2). For this volume a gradation scale of the likelihood of severity was built. Conclusion: Increase of absolute oesophageal volume included in the 95\%-isodose correlates with severity of acute esophagitis in LA-NSCLC patients treated with neo-adjuvant concurrent HA-CRT.

Background: Somatic cell nuclear transfer (SCNT) using genetically engineered donor cells is currently the most widely used strategy to generate tailored pig models for biomedical research. Although this approach facilitates a similar spectrum of genetic modifications as in rodent models, the outcome in terms of live cloned piglets is quite variable. In this study, we aimed at a comprehensive analysis of environmental and experimental factors that are substantially influencing the efficiency of generating genetically engineered pigs. Based on a considerably large data set from 274 SCNT experiments (in total 18,649 reconstructed embryos transferred into 193 recipients), performed over a period of three years, we assessed the relative contribution of season, type of genetic modification, donor cell source, number of cloning rounds, and pre-selection of cloned embryos for early development to the cloning efficiency. Results: 109 (56%) recipients became pregnant and 85 (78%) of them gave birth to offspring. Out of 318 cloned piglets, 243 (76%) were alive, but only 97 (40%) were clinically healthy and showed normal development. The proportion of stillborn piglets was 24% (75/318), and another 31% (100/318) of the cloned piglets died soon after birth. The overall cloning efficiency, defined as the number of offspring born per SCNT embryos transferred, including only recipients that delivered, was 3.95%. SCNT experiments performed during winter using fetal fibroblasts or kidney cells after additive gene transfer resulted in the highest number of live and healthy offspring, while two or more rounds of cloning and nuclear transfer experiments performed during summer decreased the number of healthy offspring. Conclusion: Although the effects of individual factors may be different between various laboratories, our results and analysis strategy will help to identify and optimize the factors, which are most critical to cloning success in programs aiming at the generation of genetically engineered pig models.

Background: Congenital heart defects (CHD) is the most common cause of death from a congenital structure abnormality in newborns and is often associated with fetal loss. There are many types of CHD. Human genetic studies have identified genes that are responsible for the inheritance of a particular type of CHD and for some types of CHD previously thought to be sporadic. However, occasionally different members of the same family might have anatomically distinct defects - for instance, one member with atrial septal defect, one with tetralogy of Fallot, and one with ventricular septal defect. Our objective is to identify susceptibility loci for CHD in families affected by distinct defects. The occurrence of these apparently discordant clinical phenotypes within one family might hint at a genetic framework common to most types of CHD. Results: We performed a genome-wide linkage analysis using MOD score analysis in families with diverse CHD. Significant linkage was obtained in two regions, at chromosome 15 (15q26.3, P-empirical = 0.0004) and at chromosome 18 (18q21.2, P-empirical = 0.0005). Conclusions: In these two novel regions four candidate genes are located: SELS, SNRPA1, and PCSK6 on 15q26.3, and TCF4 on 18q21.2. The new loci reported here have not previously been described in connection with CHD. Although further studies in other cohorts are needed to confirm these findings, the results presented here together with recent insight into how the heart normally develops will improve the understanding of CHD.

Background: To determine if black-blood 3 T cardiovascular magnetic resonance (bb-CMR) can depict differences between symptomatic and asymptomatic carotid atherosclerotic plaques in acute ischemic stroke patients. Methods: In this prospective monocentric observational study 34 patients (24 males; 70 +/- 9.3 years) with symptomatic carotid disease defined as ischemic brain lesions in one internal carotid artery territory on diffusion weighted images underwent a carotid bb-CMR at 3 T with fat-saturated pre- and post-contrast T1w-, PDw-, T2w- and TOF images using surface coils and Parallel Imaging techniques (PAT factor = 2) within 10 days after symptom onset. All patients underwent extensive clinical workup (lab, brain MR, duplex sonography, 24-hour ECG, transesophageal echocardiography) to exclude other causes of ischemic stroke. Prevalence of American Heart Association lesion type VI (AHA-LT6), status of the fibrous cap, presence of hemorrhage/thrombus and area measurements of calcification, necrotic core and hemorrhage were determined in both carotid arteries in consensus by two reviewers who were blinded to clinical information. McNemar and Wilcoxon's signed rank tests were use for statistical comparison. A p-value

Background: In recent years, personalized medicine (PM) has become a highly regarded line of development in medicine. Yet, it is still a relatively new field. As a consequence, the discussion of its future developments, in particular of its ethical implications, in most cases can only be anticipative. Such anticipative discussions, however, pose several challenges. Nevertheless, they play a crucial role for shaping PM's further developments. Therefore, it is vital to understand how the ethical discourse on PM is conducted, i.e. on what - empirical and normative - assumptions ethical arguments are based regarding PM's current and future developments. Methods: To gather this information, we conducted a qualitative interview study with stakeholders in the German health care system. Our purposive sample included 17 representatives of basic research, clinical research, health economics, regulatory authorities, reimbursement institutions, pharmaceutical industry, patient organizations, as well as clinicians and legal experts involved in PM developments or policy making. We used an interview guide with open-ended questions and analyzed transcriptions of the interviews by means of qualitative content analysis. Results: The respondents addressed a multitude of concerns in the context of research on as well as application of personalized preventive and therapeutic measures both on the individual and on the societal level. Interestingly, regarding future developments of PM the ethical evaluation seemed to follow the rule: the less likely its application, the more problematic a PM measure is assessed. The more likely its application, on the other hand, the less problematic it is evaluated. Conclusions: The results of our study suggest re-focusing the ethical discourse on PM in Germany towards a constructive ethical monitoring which ensures to include only, nevertheless all of the actual and/or potential concerns that are ethically relevant in order to allow balancing them against the actual and potential ethically relevant benefits of PM measures. To render this possible, we propose a strategy for evaluating ethical concerns in the context of PM.

UK Biobank is a prospective cohort study with 500,000 participants aged 40 to 69. Recently an enhanced imaging study received funding. Cardiovascular magnetic resonance (CMR) will be part of a multi-organ, multi-modality imaging visit in 3-4 dedicated UK Biobank imaging centres that will acquire and store imaging data from 100,000 participants (subject to successful piloting). In each of UK Biobank's dedicated bespoke imaging centres, it is proposed that 15-20 participants will undergo a 2 to 3 hour visit per day, seven days a week over a period of 5-6 years. The imaging modalities will include brain MRI at 3 Tesla, CMR and abdominal MRI at 1.5 Tesla, carotid ultrasound and DEXA scans using carefully selected protocols. We reviewed the rationale, challenges and proposed approaches for concise phenotyping using CMR on such a large scale. Here, we discuss the benefits of this imaging study and review existing and planned population based cardiovascular imaging in prospective cohort studies. We will evaluate the CMR protocol, feasibility, process optimisation and costs. Procedures for incidental findings, quality control and data processing and analysis are also presented. As is the case for all other data in the UK Biobank resource, this database of images and related information will be made available through UK Biobank's Access Procedures to researchers (irrespective of their country of origin and whether they are academic or commercial) for health-related research that is in the public interest.

Background: An association between eosinophilic esophagitis (EoE) and celiac disease (CD) has been suggested in the literature. Our aim was to confirm and quantify the association between these two diseases. Methods: All patients in a large Canadian city diagnosed with EoE or CD over a five-year period were identified. Standardized incidence ratios (SIRs) with 95\% confidence intervals (CIs) were calculated. Results: Over the five-year study EoE was diagnosed in 421 patients and CD was diagnosed in 763 patients. The incidence of EoE ranged from 2.1 to 10.7 cases per 100,000 population. The incidence of CD ranged from 10.4 to 15.7 cases per 100,000 population. Among the EoE cohort, 83 (20\%) cases of EoE and 245 (32\%) cases of CD were diagnosed in pediatric patients. The incidence of EoE in the pediatric subpopulation ranged from 3.7 to 6.9 cases per 100,000 population. The incidence of CD in the pediatric subpopulation ranged from 9.5 to 22.7 cases per 100,000 population. The concomitant diagnosis of both EoE and CD was made in three patients, all of whom were pediatric males. The SIR for EoE in the CD cohort was 48.4 (95\% CI = 9.73, 141.41) with a SIR for CD within the paediatric EoE cohort of 75.05 (95\% CI = 15.08, 219.28). Conclusions: This study confirms the association between EoE and CD. However, this association may be limited to pediatrics where the risk of each condition is increased 50 to 75-fold in patients diagnosed with the alternative condition. The concomitant diagnosis of these conditions should be considered in pediatric patients with upper gastrointestinal symptoms.

Background: Obesity is associated with abnormal fasting and postprandial lipids, which may link obesity with atherosclerosis. We explored fasting and postprandial lipids in morbidly obese patients treated with sleeve gastrectomy and in control subjects. Methods: After fasting for 12 h 15 morbidly obese patients (BMI 51.4 +/- 6.5 kg/m(2), 43.7 +/- 12.6 years) received a standardized oral fat load before and 3 months after bariatric surgery (sleeve gastrectomy). Controls (n=9, BMI 23.1 +/- 1.4 kg/m(2)) were studied once. Plasma was obtained fasting and then postprandially every 2 h for 8 h. Triglycerides (TG), chylomicron-TG (CM-TG), VLDL/chylomicron-remnant (VLDL/CR)-TG, cholesterol, LDL-cholesterol, VLDL/CR-cholesterol and HDL-cholesterol were isolated by ultracentrifugation at each time point. Postprandial values were expressed as area under the curve (AUC) and incremental area under the curve (iAUC). In addition, fasting glucose and insulin values and HOMA-IR-Index was measured (n=14). Results: Compared to controls morbidly obese patients had elevated TG and slightly altered postprandial lipids. Following surgery (weight loss 23.4 kg +/- 6.2 kg; 150 mg/dl) a similar pattern was observed. Fasting insulin and HOMA were reduced significantly (-51.9%; p=0.004 and -47.9%; p=0.011). Conclusions: Three months after sleeve gastrectomy fasting and postprandial lipoprotein metabolism and glucose metabolism is improved in morbidly obese patients. The potential mechanisms may relate to decreased caloric intake but also to hormonal changes.

Background: A key issue for safe and reproducible gene therapy approaches is the autologous and tissue-specific expression of transgenes. Tissue-specific expression in vivo is either achieved by transfer vectors that deliver the gene of interest into a distinct cell type or by use of tissue-specific expression cassettes. Here we present the generation of non-viral, episomally replicating vectors that are able to replicate in a tissue specific manner thus allowing tissue specific transgene expression in combination with episomal replication. The episomal replication of the prototype vector pEPI-1 and its derivatives depends exclusively on a transcription unit starting from a constitutively active promoter extending into the scaffold/matrix attachment region (S/MAR). Results: Here, we exchanged the constitutive promoter in the pEPI derivative pEPito by the tumor specific alpha fetoprotein (AFP) or the muscle specific smooth muscle 22 (SM22) promoter leading to specific transgene expression in AFP positive human hepatocellular carcinoma (HUH7) and in a SM22 positive cell line, respectively. The incorporation of the hCMV enhancer element into the expression cassette further boosted the expression levels with both promoters. Tissue specific-replication could be exemplary proven for the smooth muscle protein 22 (SM22) promoter in vitro. With the AFP promoter-driven pEPito vector hepatocellular carcinoma-specific expression could be achieved in vivo after systemic vector application together with polyethylenimine as transfection enhancer. Conclusions: In this study we present an episomal plasmid system designed for tissue specific transgene expression and replication. The human AFP-promoter in combination with the hCMV enhancer element was demonstrated to be a valuable tissue-specific promoter for targeting hepatocellular carcinomas with non-viral gene delivery system, and tissue specific replication could be shown in vitro with the muscle specific SM22 promoter. In combination with appropriate delivery systems, the tissue specific pEPito vector system will allow higher tissue-specificity with less undesired side effects and is suitable for long term transgene expression in vivo within gene therapeutical approaches.

Background: The MCMV major immediate early promoter/enhancer (MIEP) is a bidirectional promoter that drives the expression of the three immediate early viral genes, namely ie1, ie2 and ie3. The regulation of their expression is intensively studied, but still incompletely understood. Methods: We constructed a reporter MCMV, (MCMV-MIEPr) expressing YFP and tdTomato under the control of the MIEP as proxies of ie1 and ie2, respectively. Moreover, we generated a liver sinusoidal endothelial cell line (LSEC-uniLT) where cycling is dependent on doxycycline. We used these novel tools to study the kinetics of MIEP-driven gene expression in the context of infection and at the single cell level by flow cytometry and by live imaging of proliferating and G(0)-arrested cells. Results: MCMV replicated to higher titers in G(0)-arrested LSEC, and cycling cells showed less cytopathic effect or YFP and tdTomato expression at 5 days post infection. In the first 24 h post infection, however, there was no difference in MIEP activity in cycling or G(0)-arrested cells, although we could observe different profiles of MIEP gene expression in different cell types, like LSECs, fibroblasts or macrophages. We monitored infected LSEC-uniLT in G(0) by time lapse microscopy over five days and noticed that most cells survived infection for at least 96 h, arguing that quick lysis of infected cells could not account for the spread of the virus. Interestingly, we noticed a strong correlation between the ratio of median YFP and tdTomato expression and length of survival of infected cells. Conclusion: By means of our newly developed genetic tools, we showed that the expression pattern of MCMV IE1 and IE2 genes differs between macrophages, endothelial cells and fibroblasts. Substantial and cell-cycle independent differences in the ie1 and ie2 transcription could also be observed within individual cells of the same population, and marked ie2 gene expression was associated with longer survival of the infected cells.

Background: Cardiovascular magnetic resonance (CMR) has become an important diagnostic imaging modality in cardiovascular medicine. However, insufficient image quality may compromise its diagnostic accuracy. We aimed to describe and validate standardized criteria to evaluate a) cine steady-state free precession (SSFP), b) late gadolinium enhancement (LGE), and c) stress first-pass perfusion images. These criteria will serve for quality assessment in the setting of the Euro-CMR registry. Methods: Thirty-five qualitative criteria were defined (scores 0-3) with lower scores indicating better image quality. In addition, quantitative parameters were measured yielding 2 additional quality criteria, i.e. signal-to-noise ratio (SNR) of non-infarcted myocardium (as a measure of correct signal nulling of healthy myocardium) for LGE and \% signal increase during contrast medium first-pass for perfusion images. These qualitative and quantitative criteria were assessed in a total of 90 patients (60 patients scanned at our own institution at 1.5T (n=30) and 3T (n=30) and in 30 patients randomly chosen from the Euro-CMR registry examined at 1.5T). Analyses were performed by 2 SCMR level-3 experts, 1 trained study nurse, and 1 trained medical student. Results: The global quality score was 6.7 +/- 4.6 (n=90, mean of 4 observers, maximum possible score 64), range 6.4-6.9 (p=0.76 between observers). It ranged from 4.0-4.3 for 1.5T (p=0.96 between observers), from 5.9-6.9 for 3T (p=0.33 between observers), and from 8.6-10.3 for the Euro-CMR cases (p=0.40 between observers). The inter- (n=4) and intra-observer (n=2) agreement for the global quality score, i.e. the percentage of assignments to the same quality tertile ranged from 80\% to 88\% and from 90\% to 98\%, respectively. The agreement for the quantitative assessment for LGE images (scores 0-2 for SNR 5, respectively) ranged from 78-84\% for the entire population, and 70-93\% at 1.5T, 64-88\% at 3T, and 72-90\% for the Euro-CMR cases. The agreement for perfusion images (scores 0-2 for \% SI increase >200\%, 100\%-200\%,

Background: Patellofemoral complications are one of the main problems after Total Knee Arthroplasty (TKA). Retropatellar pressure distribution after TKA can contribute to these symptoms. Therefore we evaluated retropatellar pressure distribution subdivided on the ridge, medial and lateral surface on non-resurfaced patella before and after TKA. Additionally, we analyzed axial femorotibial rotation and quadriceps load before and after TKA. Methods: Seven fresh frozen cadaver knees were tested in a force controlled knee rig before and after TKA (Aesculap, Tuttlingen, Germany, Columbus CR) while isokinetic flexing the knee from 20 degrees to 120 degrees under weight bearing. Ridge, medial and lateral retropatellar surface were defined and pressure distribution was dynamically measured while quadriceps muscles and hamstring forces were applied. Aside axial femorotibial rotation and quadriceps load was recorded. Results: There was a significant change of patella pressure distribution before and after TKA (p = 0.004). In physiological knees pressure distribution on medial and lateral retropatellar surface was similar. After TKA the ridge of the patella was especially in higher flexion grades strongly loaded (6.09 +/-1.31 MPa) compared to the natural knee (2.92 +/-1.15 MPa, p < 0.0001). Axial femorotibial rotation showed typical internal rotation with increasing flexion both before and after TKA, but postoperatively it was significantly lower. The average amount of axial rotation was 3.5 degrees before and after TKA 1.3 degrees (p = 0.001). Mean quadriceps loading after implantation of knee prosthesis did not change significantly (575 N +/- 60 N in natural knee and after TKA 607 N +/- 96 N; p = 0.28). Conclusions: The increased retropatellar pressure especially on the ridge may be one important reason for anterior knee pain after TKA. The trochlea of the femoral component might highly influence the pressure distribution of the non-resurfaced retropatellar surface. Additionally, lower axial femorotibial rotation after TKA might lead to patella maltracking. Changing the design of the prosthesis or a special way of patella shaping might increase the conformity of the patella to trochlea to maintain natural contact patterns.

Background: Prostate smooth muscle tone is regulated by alpha 1-adrenoceptor-induced contraction and cAMP-mediated relaxation. EPAC is an effector of cAMP, being involved in smooth muscle relaxation and cell cycle control outside the lower urinary tract. Here, we investigated the expression and function of EPAC in human prostate tissues from patients undergoing radical prostatectomy. Results: mRNA and protein expression of EPAC was detected in all prostate tissues by RT-PCR and Western blot analysis. Immunoreactivity was observed in stromal cells, and colocalized with immunofluorescence for a-smooth muscle actin and calponin. Under normal conditions, noradrenaline-or phenylephrine-induced contraction of prostate strips in the organ bath was not affected by the EPAC activator pCPT (SP-8-pCPT-2'-O-Me-cAMPS.NA) (30 mu M). However, when the cyclooxygenase inhibitor indomethacin (50 mu M) was added, EPAC activators pCPT and OME (8-CPT-2'-O-Me-cAMP.Na) (30 mu M) significantly reduced contractions by low concentrations of phenylephrine. These effects were not observed on noradrenaline-induced contraction. OME and pCPT caused phosphorylation of the transcription factor Elk1 in prostate tissues. Elk1 activation was confirmed by EMSA (electrophoretic mobility shift assay), where OME and pCPT incresed Elk1 binding to a specific DNA probe. Conclusions: EPAC activation may reduce alpha 1-adrenergic prostate contraction in the human prostate, although this effect is masked by cyclooxygenases and beta-adrenoceptors. A main EPAC function in the human prostate may be the regulation of the transcription factor Elk1.